Toxicity of Nanoscaled Zero-Valent Iron Particles on Tilapia, Oreochromis mossambicus

This research effort aims to evaluate the hazardous potential of the redox state (OH–) of zero-valent iron nanoparticles (nZVI) and its histopathological and oxidative stress toward Mozambique tilapia, Oreochromis mossambicus. X-ray powder diffraction (XRD) validated the nZVI nanoparticles’ chemical composition, while transmission electron microscopy (TEM) revealed that their physical form is round and oval. The exposure to 10 g/mL of nZVI induced the activation of the cellular superoxide dismutase (SOD) activity. Dose-dependent testing of O. mossambicus had a reduction in SOD and an increase in malondialdehyde (MDA) levels, suggesting that nZVI caused oxidative damage. At a concentration of 100 g/mL, the catalase (CAT) and peroxidase (POD) activities of diverse tissues exhibited a gradual decrease after 2 days of exposure and a fast increase until day 6. The scavenging of reactive oxygen species (ROS) in the epidermis, liver, and gills of O. mossambicus deteriorated and accumulated gradually. MDA levels in the skin, gill, and liver tissues were substantially higher after 8 days of exposure to 100 and 200 g/mL nZVI compared to those of the control group and those exposed to 10 and 50 g/mL nZVI for 2 days. Extreme histological and morphological abnormalities were seen in the skin, gill, and liver tissues of experimental animals, demonstrating that the damage resulted from direct contact with nZVI in water. A one-way ANOVA followed by Dunnett’s post-test was performed to investigate significant differences.


■ INTRODUCTION
The properties of nanoparticles that contribute to biological perturbations strongly depend on their size, mineralogy, crystallinity, and surface reactivity, which is directly connected to nanoparticle toxicity through redox reactions, the production of oxygen-or nitrogen-free radicals, the dissolution of nanoparticles, the release of toxic ions, and the sorption and transport of metal ions or xenobiotic pollutants. 1 There is an understood assumption that zero-valent iron nanoparticles (nZVI) are relatively nontoxic because Fe 0 simply oxidizes to Fe 2+ and then to Fe 3+ , both of which are common chemical species in the environment that most organisms are well adjusted. However, the usage of nZVI applications increases the concentration of Fe 2+ and/or Fe 3+ substantially at a local level in the short term. nZVI oxidation can also contribute to the production of reactive oxygen species (ROS), such as hydroxyl radicals (OH − ) from superoxide (O 2 − ) and hydrogen peroxide (H 2 O 2 ) in living cells. 2−4 There are reports on the toxic effects of iron nanoparticles. Previous studies have reported the cytotoxic effects of iron oxide nanoparticles on the cytoskeleton of growing neurons and human melanoma cells. 5,6 Recent studies showed that uncoated nZVI produced neurotoxic in cultured neurons, whereas nZVI surface modified with polyaspartate decreased nanoparticle (NP) toxicity by reducing sedimentation, which limited the cell's exposure to particles. 7 The highly redox-reactive iron NPs may migrate to the surface water, penetrate plants and animals, induce toxic effects, and persistently accumulate in the ecosystem. 8 Li et al. 9 revealed the dose-dependent oxidative damage to disturbed antioxidant balance, lipid peroxidation, and morphological alterations in Medaka fish (Oryzias latipes) at embryonic or mature stages after 2 weeks of aqueous exposure to a commercial iron NP. The different toxicokinetics and dynamics between cells and an intact organism and understanding the in vivo toxic effects of nZVI and associated iron nanoparticles are important.
Iron is involved in the cellular respiration of animals and photosynthesis in plants and is an integral cofactor of ribonucleotide reductase. However, excess iron is toxic, acting as a catalyst in the Fenton reaction generating free radicals. In particular, the biodistribution and toxicity to environments with different concentrations of nZVI in environmental media have not been studied in detail. The toxic effects of nZVI on Oreochromis mossambicus were studied using histopathological and antioxidative stress markers such as superoxide dismutase, catalase, and peroxidase activities to determine the potential effects on oxidative stress and antioxidant defense, and the level of lipid peroxidation was measured for the content of malondialdehyde induced by nZVI. The synthesized nZVI was characterized using X-ray diffraction, field-emission scanning electron microscope, and transmission electron microscope leading to the addressing of the toxicity of nZVI.
Synthesis of nZVI. The method for the synthesis of nZVI was performed as reported by Kanel et al. 10 The deoxygenated water is used throughout the experimentation. Five grams of FeSO 4 .7H 2 O was dissolved in 250 mL of 30% cold ethanol, and an appropriate amount of NaBH 4 was added to the above solution dropwise with a drop rate of 5 mL/min using a peristaltic pump (Rivotek-India) under stirring at 40.32g. The solution slowly turned black color indicating the formation of nZVI. Thus, the formed nZVI was centrifuged at 280g, washed thrice with ethanol, and then dried and pulverized. The temperature was maintained at less than 20°C.
Fish. Tilapia fish (O. mossambicus) of mean weight 35.46 ± 2.3 g/wet/wt. were purchased from a local fish farm (Raam Raghu Fish Farm, Walajapet, Tamil Nadu, India). They were kept in a 300 L fiberglass tank with recirculating water for 3 weeks (22 ± 0.5°C, 12 h light:12 h dark cycles). The fish were fed once every other day with commercial fish food (J.W. Vitra, 35% protein).
Tissue Preparations. At the end of the experimental duration of 8 days, the fish of control and test groups were sacrificed by decapitation. Gills, liver, and skin of control and treated fish were dissected out, fixed in Bouin's solution for 24 h, and then were processed for the paraffin (m.p. 62°C) embedding procedure. 11 Histopathology. Paraffin blocks of gills, liver, and skin of all of the groups were sliced at a 6 μm thickness and stretched on sterilized glass slides. After deparaffinization, sections were stained with hematoxylin−eosin and observed under light microscopy. The histopathological changes in the tissues were examined in the randomly selected 10 sections from each fish. The average occurrence of each histopathological parameter was categorized as mild (+, <25% of the sections), moderate (++, 25−50% of the sections), and severe (+++, >50% of the sections). Histopathological changes induced by treatments in the tissues were photographed using a Digi 3 compound binocular microscope (Labomed, USA) fitted with a photomicrographic attachment. 12 Characterization of Synthesized nZVI. The synthesized nZVI was characterized for size and dispersity before exposure to O. mossambicus. This is accomplished using transmission electron microscopy (TEM, JEOL, model 1200 EX). Iron nanoparticles were placed in a glass holder and scanned from 20 to 60°. This scan range covered all major species of iron and iron oxide. The scanning rate was set at 2.0°/min. Lanthanum hexaboride (LaB6) was used to calibrate the instrument before analysis. The X-ray powder diffraction (XRD) analysis was conducted with an XRD 3100 diffractometer (Phillips Electronic Co., Eindhoven, Netherlands) at 45 kV and 30 mA. TEM measurements were operated at an accelerating voltage of 120 kV and later with an XDL 3000 powder. ■ EXPERIMENTAL SECTION Acute Toxicity. Adult fish (10 fishes per group of concentrations) were maintained in 10 L glass aquaria and exposed to a graded series of nZVI in control (FeSo 4 ), 10, 50, 100, and 200 μg/mL for 8 days. Triplicate was performed for each concentration. 13 Fish were subjected to acute toxicity tests, and the control fish were not fed during the experimental period. The experimental fish from each group were sacrificed in ice−water, dried with filter paper, weighed, and finally anatomized for the collection of the test tissues including skin, gill, and liver. 14,15 Water Analysis. Water samples were collected directly before and after each water change for pH (YSI 63 pH meter), total ammonia (HI 95715, Hanna Instruments), and oxygen saturation (YSI 85 D.O. meter). Water used showed a conductivity of less than 1 μV/cm and either total organic carbon (TOC) less than 2 mg/L or chemical oxygen demand (COD) less than 5 mg/L. There were no treatment differences in water quality between tanks (ANOVA, p > 0.05).
Biochemical Tests. Total Glutathione (GSH) Levels. The liver tissues from the test fish were homogenized in ice-cold 5% sulfosalicylic acid and centrifuged for 10 min at 4°C, after which the supernatant was incubated with 6.3 mM of Na-EDTA, 6 mM of dithionitrobenzoic acid, 0.25 mg/L of NADPH, and 1 unit/mL of GSH reductase in 143 mM of sodium phosphate (pH 7.5) at room temperature for 6 min. The absorbance was then measured at 405 nm. 16,17 Protein Assay. The protein content of each sample was determined using bovine serum albumin as a standard. 18 Preparation of Tissues for Metal Analysis by Atomic Absorption Spectrophotometry. Tissue samples were digested using HNO 3 (4 mL per gram tissue) at 70°C on a hot plate until NO 2 evaporation ceased. 19 A volume of reagent-grade 10% H 2 O 2 equal to the initial HNO 3 was added to the digested samples until the sample became clear and then allowed to cool to an ambient temperature. After cooling, the solution was filtered and the filtrate made up to a known volume (100 mL) with deionized water. The samples were stored cool at 4°C till the metals were analyzed. All solutions were analyzed using a Varian Spectra AA240. The variability of the metal determinations was assessed using Standard Reference Materi-al (1566A oyster tissue; National Institute of Standards and Technology, USA). The values are expressed as μg/mL on a wet-weight basis for fish tissue samples.
Oxidative Stress Parameter Analysis. O. mossambicus was exposed to 0, 10, 50, 100, and 200 μg/mL of nZVI for 8 days using a semistatic exposure test. The experiment was designed to allow for acute physiological effects over the exposure period. Five fish per treatment were randomly collected on days 1, 2, 4, 6, and 8, respectively, for biochemical analysis. Skin, gill, and liver tissues were removed separately and immediately snap-frozen in liquid nitrogen and stored at −20°C until needed. The frozen tissues were rinsed in 9-fold chilled 100 mmol/L, pH 7.8 sodium phosphate buffer solution and homogenized by a hand-driven glass homogenizer. The homogenates were centrifuged at 11 200g at 4°C for 20 min, and the supernatant was stored in Eppendorf tubes at 4°C. The liver supernatant was diluted with a 9-fold chilled sodium phosphate buffer solution to 1%. The prepared supernatants were analyzed for antioxidant enzymes, i.e., superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) activities, to determine possible effects on oxidative stress and antioxidant defense, and the lipid peroxidation (LPO) level was measured for the content of malondialdehyde (MDA). All assays were performed in triplicate. The SOD (EC 1.15.1.1) activity was estimated based on its ability to inhibit the reduction of nitrobluetetrazolium (NBT) by superoxide radicals generated by xanthine/xanthine oxidase according to the modified method of Beauchamp and Fridovich. 20 One unit of SOD activity could be defined as the quantity of SOD required to produce a 50% inhibition of NBT reduction under the experimental conditions, and the specific enzyme activity was expressed as units per gram fresh weight of tissue per hour. The CAT (EC 1.11.1.6) activity was determined using the method of measuring the initial rate of the decrease in absorbance at 240 nm as a consequence of H 2 O 2 consumption over 1 min. The activity was expressed as a unit per gram fresh weight of tissue. 21 The POD (EC 1.11.1.7) activity was assayed using guaiacol as a hydrogen donor by measuring the change at 470 nm over 1 min, as reported previously. 22 Enzyme activity was defined as a unit (one activity unit defined as absorbance at 470 nm changes 0.01 per min) per gram fresh weight of tissue. LPO (EC 1.11.1.11) was measured using the thiobarbituric acid (TBA) assay (Willmore and Storey). 21 The chromogen formed was measured by fluorometry. 23 The level of LPO was expressed as μmol MDA/g fresh tissue.
Statistical Analysis. All experiments were repeated three times independently. Data were recorded as the mean with the standard deviation. For antioxidant assays, the statistical differences were analyzed with one-way ANOVA followed by Dunnett's post-test, which was used to detect significant differences between the control and treated groups using Graph Pad Prism software 6.0 for Windows, Graph Pad Software, San Diego, California, USA, www.graphpad.com. There was no significance in the day's dependent values. p < 0.05 was considered statistically significant. The no observed effect concentration (NOEC) value was designated as the highest tested concentration that had no statistically significant effect within the exposure period when compared with the control.
■ RESULTS AND DISCUSSION Characterization. The powder XRD pattern of the synthesized nZVI is shown in Figure 1A. The XRD pattern showed an intense peak at 44.71 and a less intense peak at 65.54, which could be assigned to (110) and (200) of cubic Fe, respectively, and agreed with the database of the joint committee on powder diffraction standards (JCPDS No. 00-006-0696). The TEM study revealed the shape of the nZVI to be spherical and oval ( Figure 1B,C). The selected area diffraction pattern (SAED) reveals that the particles were amorphous in nature and the patterns indexed as (110), (200), and (211) reflections of the elemental characterization were made by EDX ( Figure 1D,E). The size was found to be in a range of 60−80 nm, which was consistent with the XRD graph using Scherrer's constant 24 (Figure 2). Protein Estimation. According to the Bradford method, the protein estimation was found to be changed in both the test and control ( Figure 3A). The calorimetric value obtained yields low control values for the tissue samples employed in the presence of nZVI, hence giving an immediate reduction in the optical density.
Estimation of the Fe (nZVI) Content in Tissues by Atomic Absorption Spectrophotometry. The data indicate that the O. mossambicus retains regulatory mechanisms to increase Fe availability by regulating the gill Fe(III) iron acquisition, which might depend on a high cellular Na/K gradient. This view further implies that gills could act as a route and site for the Fe (nZVI) iron uptake and its subsequent accumulation during continuous exposure to metal contamination ( Figure 3B). The gills are important in the uptake and loss of the major body electrolytes, acid−base equivalents, ammonia, respiratory gases, and many waterborne contaminants. Some of these flux parameters are relatively easy to measure and have been applied to metal toxicity studies with fish and crustaceans. The Fe(III) iron content for the skin and liver tissue has been shown to be affected the least. The Certified Standard Reference Material SRM 1566a (oyster tissue) from NIST, USA, was used to verify the procedure, yielding good agreement between the measured and verified concentrations (±10%).
Histopathology. The fish exposed to 20 and 40 μg/L nZVI exhibited irregular performance like erratic turning and loss of control. They become sluggish, and a slimy material was secreted from the whole body. The exposed fish swam to the surface more frequently than the control fish. Neither mortality nor any visible changes in behavior were observed in the control group. Tissue damages brought about by waterborne pollutants can be easily observed because the fish gills come into immediate contact with the environment. In fish, the internal environment is separated from the external environment by only a few microns of delicate gill epithelium and thus the bronchial function was very sensitive to environmental contamination. 25 The gill of O. mossambicus made up of primary lamellae was arranged in double rows, projecting on the lateral sides of which are a series of alternately arranged secondary lamellae. At the core, there is a cartilaginous supporting rod and blood vessels with traces of sinusoidal blood spaces. No histopathological changes were pragmatic in the gill and liver of the control fish. The structural details of the   Figure 4. Histopathological results designated that the gills are the primary target tissue affected by nZVI. The most mutual changes at all amounts of nZVI were desquamation and necrosis. At day 1 of exposure to 10, 50, 100, and 200 μg/mL nZVI, the gills of test fish showed aneurism in many areas of secondary lamellae with the breakdown of the pillar cell system. In the acute nZVI exposed fish lamellar fusion, epithelial lifting, desquamation, aneurism, and curling of secondary lamellae were detected. The gills were swollen in comparison to the control fish because of the hypertrophy and hyperplasia of the gill epithelial cells. The proliferative changes in the epithelium of gill filaments and secondary lamellae degenerative and necrotic changes in gill filaments were observed. In addition, the separation of the epithelium of the secondary lamellae from the lamellar supporting cell in gill filaments, intravascular hemolysis, and dilation in the blood vessels of gill filaments, hemorrhage between gill filaments, edema in secondary lamellae, and mucus accumulation between gill filaments were seen. Edema, desquamation, fusion of secondary lamellae, and necrosis of lamellar epithelium were observed. In 100 μg/L, group aneurism and curling of secondary lamellae were more prominent. The lifting of the lamellar epithelium and edema was perceived after 2 and 4 days of exposure to 50 μg/mL and day 4 of exposure to 100 μg/mL. Epithelial hyperplasia and fusion of the secondary lamellae were observed after days 4−6 of exposure to 100 μg/ mL of nZVI and day 8 of exposure to 200 μg/mL of nZVI ( Figure 4B,C).
The skin comprising the general body surface of the fish is often considered a robust barrier to the external environment, and like that of mammals, it consists of three layers: the epidermis, the dermis (scales in the case of fish), and the hypodermis. The normal skeletal muscles are composed chiefly of segmental myomeres. Each myomere is regarded as an apparent muscle and its fibers are parallel to the long axis of the body. In the epithelium of the caudal, part the numerous mucous cells were displaced by the epithelial cells toward the basal membrane, producing the appearance of a single-layered epithelium. The small fibers were stained lightly, and most of the intermediate fibers were moderately stained, although a few intermediate fibers were also intensely stained. The structural details of the skin of the control of O. mossambicus are shown The fish were not exposed to nZVI, showing a cartilaginous core, primary lamellae, and secondary lamellae exposed to (B) 100 μg/mL of nZVI and (C) 200 μg/mL of nZVI, depicting lamellar aneurism, cellular necrosis, and vacuole formation. The fish were not exposed to nZVI, (B) exposed to 100 μg/mL of nZVI, and (C) exposed to 200 μg/mL of nZVI, which are shown to have cellular damage. The fish were not exposed to nZVI. The fish were exposed to (B) 100 μg/mL of nZVI and (C) 200 μg/mL of nZVI.
in Figure 5A−C. The vacuole formation and necrosis of the skin cells were found in 50 and 100 μg/mL, respectively.
Liver is a sensitive organ and an important nucleus of material metabolism in animals. The liver carries out the biotransformation of exogenous xenobiotics predominantly. The significant reduction of liver weight to body weight ratio is also observed in O. mossambicus treated with different concentrations of nZVI. The ratio is increased in a concentration-dependent manner. The liver histology of O. mossambicus exhibited a parenchymal architecture of the hepatocytes. The hepatocytes contain homogeneous cytoplasm with a centrally placed nucleus ( Figure 6A−C). In the liver, vacuolar degeneration, focal areas of coagulative necrosis, focal areas of necrosis, destruction of hepatoportal blood vessels, and hemorrhage between the hepatocytes were observed. Besides, intravascular hemolysis and dilation were seen in hepatic and hepatoportal blood vessels. In addition, dilation and congestion were noticed in blood sinusoids. In the test group, the cytoplasmic vacuolization was prominent; lateralization and condensation of the nuclei were also observed. In 200 μg/mL, group shrinkage of hepatocytes with increased sinusoidal blood spaces was observed ( Figure 6B). The hepatocytes more frequently showed pyknotic nuclei in this group. The alterations in liver due to toxicity impact are often associated with a degenerative necrotic condition. The changes induced by nZVI in the liver hepatocytes such as vacuolization, necrosis, and nuclear condensation were found after exposure to nZVI. The scientific community has carried out many experimentations toward the toxicity of nanoparticles on various animal models from Saccharomyces, 26,27 Drosophila, 28,29 Caenorhabditis elegans, 30,31 zebrafish, 32,33 and many more.
The pelargonidin-loaded poly-lactide-co-glycolide nanoparticles were studied to analyze the cypermethrin toxicity model of O. mossambicus and L6 muscle cell line, which confirmed the pelargonidin protective ability in fish muscle cells, as measured by percent cell viability, DNA damage, and stress-related enzymes. 34 In another study, the copper nanoparticle toxicity model was studied, specifically the olfactory mucosa of Rainbow trout, which confirmed that neither oxidative stress nor apoptosis was triggered by Cu 2+ or CuNPs in mucosal cells. 35 Behavioral Changes. The behavior of the control group fish showed normal specific behavior during the test period. In the nZVI toxicity test, the behavioral response of tilapia was conducted every 12 h. The changes in the behavioral response started 6 h after dosing. Observed behavioral changes were rapid gill movements, loss of equilibrium, spiral swimming, and staying motionless at a certain location generally at the midwater level for prolonged periods. The highest concentration of 200 mg/L showed all responses at high intensities: the loss of equilibrium motionlessness, increase in ventilation, efforts to swallow air from the water surface, and spiral swimming. 36 Oxidative Stress. The diminishing of total GSH in the liver has been related to zero increase in LPO, signifying that the liver uses up antioxidant defenses to prevent oxidative stress. The initiation of ROS was correlated with the amount of nZVI, which entered the fish cells (Figure 7). Decreased levels of GSH were also shown in the nanoparticle-treated group in a Figure 7. Total GSH level activity in liver, gill, and skin tissues of tilapia after exposure to synthesized nZVI (expressed in U/g min vs exposure time (days)). Figure 8. Total SOD level activity in liver, gill, and skin tissues of tilapia after being exposed to synthesized nZVI (expressed as U/g min vs exposure time (days)).
concentration-dependent manner in liver. Among the tissue damages found, liver is the most affected part exposed to nZVI.
SOD is the chief enzyme to deal with oxyradicals and is responsible for catalyzing the dismutation of highly superoxide radical O 2 − to O 2 + and H 2 O 2 . It is very subtle to the stress of pollutants and can be used as an oxidative stressed signal for the early warning of environmental pollution. In the present study, the SOD activities in the skin, gill, and liver tissues of O. mossambicus were exposed with a concentration and an exposure time (Figure 8). On exposure to 10 μg/mL nZVI, SOD activities of dissimilar tissues were enthused and showed a remarkable increase, which must be due to the synthesis of new enzymes or the enhancement of pre-existing enzyme levels under minor concentrations. All of the tissues were affected by the synthesized nZVI due to the increased synthesis to cope with the superoxide radicals.
CAT and POD are also the foremost enzymes in antioxidant defense systems to convert the resulting free radicals H 2 O 2 to water and oxygen. In the present study, CAT and POD activities in different tissues of O. mossambicus with a concentration and an exposure time (0, 10, 50, 100, and 200 μg/mL for 8 days) were plotted, respectively, as shown in Figures 9 and 10. On exposure to 10 μg/mL of synthesized nZVI, the CAT activity of different tissues showed a minor Figure 9. Total CAT level activity in liver, gill, and skin tissues of tilapia after exposure to synthesized nZVI (expressed in U/g min vs exposure time (days)). Figure 10. Total POD level activity in liver, gill, and skin tissues of tilapia after exposure to synthesized nZVI (expressed in U/g min vs exposure time (days)). Figure 11. Total MDA level activity in liver, gill, and skin tissues of tilapia after exposure to synthesized nZVI (expressed in U/g min vs exposure time (days)).

ACS Omega
http://pubs.acs.org/journal/acsodf Article decrease up to day 2 and then a remarkable increase was observed. Results indicated that under stress CAT activity was inhibited, and ROS scavenging weakened and accumulated gradually in the major tissues of O. mossambicus. In addition, the CAT and POD activities in liver were 2-to 3-folds and 5to 10-folds of that in gill and skin at the same exposure concentration, respectively. LPO can be defined as the oxidative deterioration of cell membrane lipids and has been used extensively as a marker of oxidative stress. In this study, MDA contents in the skin, gill, and liver tissues were not obviously different from those in control over exposure to 10 and 50 μg/mL nZVI; however, the significant increase in the MDA level was found after 8 days of exposure to 100 and 200 μg/mL of nZVI ( Figure 11). It indicated that these tissues were undergoing oxidative stress, which was consistent with our results of a higher concentration of nZVI exhibiting more potent effects of disturbance to the antioxidant defense systems in O. mossambicus.
The Mozambique tilapia fish tank water for the experiment used in the present study is not the same as a real aquatic environment (such as lakes and rivers), and in a real environment nZVI can act differently. Keller et al. 37 reported that nanoscale titanium dioxide particles (nTiO 2 ), nZnO, and cerium oxide particles (nCeO 2 ) were relatively stable in natural freshwater: over 90% of these nanomaterials remained in the water body even after standing for 6 h at a concentration of 200 mg/L. Kadaŕ et al. 38 examined the aggregation and sedimentation of nFe 2 O 3 in natural seawater: ≤30% of nFe 2 O 3 remained in the seawater after 12 h. The aggregation and sedimentation of nFe 2 O 3 may lead to a highly localized concentration. On examining the direct adherence/adsorption of nFe 2 O 3 aggregates on the embryo surface, there could be high levels of free iron ions in the exposed tissue. This iron overload could thus have toxic implications as an excessive accumulation of nFe 2 O 3 . In particular, it could lead to an imbalance in homeostasis and aberrant cellular responses, including cytotoxicity, DNA damage, oxidative stress, epigenetic events, and inflammatory processes, which would eventually lead to the observed toxicity. 39 In another study, ionic silver and nanosilver were evaluated for their involvement in controlling oxidative stress in rainbow trout (Oncorhynchus mykiss) intestinal cell lines (RTgutGC), indicating that silver inhibits selenoenzymes and does not induce oxidative stress in RTgutGC cells. 40 Nanotechnology implies research and development undertaken with particle sizes in the 1−100 nm range. Owing to their composition, small size, and shape, nanomaterials display novel properties that have diverse applications in the biomedical, electronics, and environmental fields. 41 Metal nanoparticles possess unique properties due to their size, shape, surface structure, aggregation characteristics, and chemical composition that differs from their respective soluble metal. However, water chemistry such as salinity and pH influences the toxicity of the nanoparticle investigated but possibly affects the size and shape of the particles. 42 Chen et al. 43 reported on the mitigation of the bactericidal activity of nZVI toward Escherichia coli and Bacillus subtilis in the presence of Suwannee River humic acids.
Aquatic hypoxia (oxygen depletion), leading to mass mortality of aquatic organisms, is a global concern. Hypoxia has been recently demonstrated as an endocrine disruptor because it can alter the normal reproduction and development of organisms and eventually change the ecosystem structure and function. 44 Although iron is an essential element for many organisms, excess uptake of Fe(II) causes oxidative damage to the body via redox cycling of iron and internal ROS generation, thus leading to cell death and acute toxic effects. 15,45−47 More recently, Nations et al. 48 have reported that iron oxide (Fe 2 O 3 ) NPs decreased the snout−vent length (SVL) of Xenopus laevis tadpoles at concentrations as low as 0.001 mg/L. The SVL increased at 1 mg/L of Fe 2 O 3 NPs and then steadily decreased at higher concentrations (10, 100, and 1000 mg/L). The total body length of X. laevis tadpoles exposed to 1000 mg/L of Fe 2 O 3 NPs was also significantly reduced (p ≤ 0.033) compared with that of controls.
Acute toxicity for fish refers to mechanisms that are operative in causing lethality at concentrations effective in 96 h of tests, whereas chronic toxicity refers to mechanisms causing pathology or performance decrements in trials lasting 21−30 days. 13 Gills are important in extracting oxygen from ambient water and are priority organs in xenobiotic exposure. It is known that fish take up xenobiotics through gills. Redoxactive particles encountered by the gills must therefore induce antioxidant enzyme production and consume total glutathione (GSH) levels. The gills, which generally comprise over 50% of the surface area of the fish, are in intimate and continuous contact with the external water and are the primary target. At high enough concentrations, virtually all toxicants elicit profound morphological changes in the gills caused by an acute, generalized inflammatory response. This results in rapid death by suffocation due to edematous swelling, cellular lifting, and necrosis, lamellar fusion, greatly increased water-to-blood diffusion distance, and impeded blood and water flow through and across the respiratory lamellae. 49−51 It is well known that the gills are important respiratory organs and participate in many physiological activities, including metabolite excretion, body fluid permeability balance, and acid−base regulation balance, which were vulnerable to water pollution. NPs in the liquid phase could present either a respiratory or dietary exposure risk. The gill or gut surface of fish consists of the bulk fluid (river water, gut luminal fluid), which contributes to the formation of an unstirred layer over the epithelium. 52,53 The liver is the vital organ of detoxification. The alterations in liver due to toxicity impact are often associated with a degenerative necrotic condition. The changes induced by chromium in the liver hepatocytes such as vacuolization, necrosis, and nuclear condensation were also reported for copper exposure. 54,55 The ferrous ions in cyanobacterial cells react with oxygen and hydrogen peroxide via Fenton-like reactions that produce reactive oxygen resulting in severe cell damage. Furthermore, at the pH and redox potential encountered in the cytoplasm, the importation of large quantities of Fe(II) would result in the massive precipitation of iron(III) hydroxide nanoparticles and secondary cell destruction. 15 The application of nanotechnology licenses the modification of the fundamental physical and chemical properties of conservative materials as their size is reduced to the nanoscale, offering new materials with unique electrical, optical, and mechanical properties. The C70 nanoparticles were tested for behavioral impairments and oxidative stress in the brain, muscle, and gill of adult zebrafish and showed alterations in several neurobehavior parameters of fish, indicating a clear role of nanoparticles in the toxicity of small animals. 56 The zinc oxide nanoparticles were also tested for the oxidate stress in freshwater teleost fish. 57 Another study, the oxidative stress and bioaccumulation of aluminum nanoparticles, was tested, showing that 50 μg/L −1 of aluminum ACS Omega http://pubs.acs.org/journal/acsodf Article nanoparticles caused significant oxidative damage in the liver and gill of common carp. 58 SOD, an enzymatic antioxidant, is an important line of defense against ROS. SOD is essential for the vitality of mammalian cells and plays an important role in detoxification of superoxide anions to (hydrogen peroxide) H 2 O 2 , which was further removed by the CAT enzyme. Therefore, both SOD and CAT provide a major defense against oxidative damage from ROS. Engineered nanoparticles stimulate the production of ROS in organisms and cause damage in possibly every cell component. These ROS oxidize double bonds of fatty acids in cell membrane resulting in increased permeability, rendering it more susceptible to osmotic stress. Engineered nanoparticles like TiO 2 , with photocatalytic properties upon exposure to UV light, generate ROS and can nick supercoiled DNA. 59 Zhu et al. 60 reported the generation of oxidative stress in the gills of adult Fathead Minnow (Pimephales promelas) upon exposure to nC 60 fullerene prepared with water stirring.
A number of microbial populations used for microbial degradation may also be affected by nZVI introduction, including dehalorespirers, dissimilatory iron reducers, methanogens, and homoacetogens. 61 It could conclude that the analytical approaches to NPs in the aquatic environment are still in an initial phase of development. This study demonstrates that O. mossambicus adult fish were used as a quick high-throughput, highly efficient, cost-effective, and a sensitive platform for investigating the toxicity of nZVI by means of histopathological and antioxidative stress. Their optimization is a key point to allow field experiments and monitoring programs, the latter forming the basis of a genuine risk valuation. The formation of masses in water offers the chance for other organic materials, including toxicants, to become associated with the aggregates, which will change the bioavailability of these materials and create additional toxicological concerns.

■ CONCLUSIONS
In conclusion, we have shown the emerging impact of nZVI; growing concerns have arisen about their unintentional health and environmental impact. The main objective of our report was to characterize the toxic effects of nZVI in O. mossambicus. To evaluate these effects, we characterized the synthesized nZVI using chemical and physical characterizations such as XRD, Fourier-transform infrared spectroscopy (FTIR), TEM, and SAED. The percentage of the Fe(III) iron acquisition was studied by atomic absorption spectrophotometry. Furthermore, we also investigated several biomarkers related to oxidative stress superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) activities to determine possible effects on oxidative stress and antioxidant defense, and the lipid peroxidation (LPO) level was measured for the content of malondialdehyde (MDA) induced by nZVI exposition.